T-cell-mediated cytotoxicity has been generated by culturing immune spleen cells from untreated donors without the addition of any known cellular antigens. The effectors generated preferentially lysed H-2 matched targets, but also lysed to some extent allogeneic target cells. High (H-2d) and low (H-2k,b) responder strains were identified, and strong responsiveness was a dominant characteristic. Specificity studies suggested that the effector cells were sensitized against components contained in fetal calf serum, but not in horse serum. Enhanced responses could not be obtained by preinjecting mice with irradiated syngeneic cells which raised the possibility that the response is dependent on natural killer cells.